Mono pyranopterin-containing sulfite-oxidizing enzymes (SOEs), including eukaryotic sulfite oxidases (SOs) and homologous prokaryotic sulfite dehydrogenases (SDHs), are molybdenum enzymes that exist in almost all forms of life where they catalyze the direct oxidation of sulfite into sulfate, playing a key role in protecting cells and organisms against sulfite-induced damage. To decipher their catalytic mechanism, we have previously provided structural and spectroscopic evidence for direct coordination of HPO42- to the Mo atom at the active site of the SDH from the hyperthermophilic bacterium Thermus thermophilus (TtSDH), mimicking the proposed sulfate bound intermediate proposed to be formed during catalysis. In this work, by solving the X-ray crystallographic structure of the unbound enzyme, we resolve the changes in the hydrogen bonding network in the molybdenum environment that enable the stabilization of the previously characterized phosphate-adduct. In addition, the electron paramagnetic resonance (EPR) spectroscopic study of the enzyme over a wide pH range reveals the formation of pH-dependent Mo(V) species, a characteristic feature of eukaryotic SOEs. The combined use of HYSCORE, H2O/D2O exchange and density functional theory (DFT) calculations allows the detailed characterization of a typical low pH Mo(V) species previously unreported in bacterial SOEs, underlining the conservation of the active site properties of SOEs irrespective of their source organism.